The present invention relates to a novel anilide compound and a pharmaceutical composition containing the same. More specifically, the invention relates to a compound represented by the general formula I: 
represents a divalent residue of benzene with a substituent(s), heterocycle-condensed benzene which may or may not have a substituent, pyridine which may or may not have a substituent, cyclohexane or naphthalene or 
Ar represents an aryl group which may or may not have a substituent;
X represents xe2x80x94NHxe2x80x94, oxygen atom or sulfur atom;
Y represents xe2x80x94NR4xe2x80x94, oxygen atom, sulfur atom, sulfoxide or sulfone;
Z represents single bond or xe2x80x94NR5xe2x80x94;
R4 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent;
R5 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent; and
n represents an integer of 0 to 15;
a salt thereof or a solvated compound thereof and pharmaceutical compositions comprising these compounds.
Following the transfer of the (Japanese) dietary life to European-style diets comprising high calorie and high cholesterol due to the improvement of the living standard and the increase of the ratio of aged people in the (Japanese) population, hyperlipidemia and arteriosclerotic diseases caused by hyperlipidemia have increased in number rapidly in recent years. The increase of these diseases is now one of the social problems (in Japan). Conventional pharmaceutical treatment of hyperlipidemia and arteriosclerosis has mainly targeted the reduction of lipid in blood as the etiology thereof. The treatment has never targeted arteriosclerotic lesions of themselves. Acyl coenzyme A cholesterol acyltransferase (ACAT) is the enzyme to catalyze the synthesis of cholesterol ester from cholesterol to play a significant role in the cholesterol metabolism and absorption in gastrointestinal tract. It is suggested that the inhibition of ACAT esterifying free cholesterol in the epidermal cell of small intestine works to inhibit cholesterol absorption from intestinal lumen and that the inhibition of cholesterol ester generation in liver owing to ACAT inhibition suppresses VLDL secretion from liver into blood stream, with the resultant action to decrease blood cholesterol. It is considered that many of conventional ACAT inhibitors function as anti-lipidemia agents to exert the action of decreasing blood cholesterol by allowing the inhibitors to react with the ACAT enzyme in small intestine and liver.
As ACAT inhibitors, for example, U.S. Pat. No. 4,716,175 describes 2,2 dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide; and EP 372, 445 describes Nxe2x80x2-(2,4-difluorophenyl)-N-[5-(4,5-diphenyl-1H-imidazol-2-ylthio)pentyl]-N-heptyl urea in the specification. However, many of these conventional ACAT inhibitors as anti-hyperlipidemia agents principally work to decrease blood cholesterol and are administered at large doses to permit sufficient exertion of the action. Due to the emergence of side effects including intestinal bleeding, intestinal disorders, diarrhea and liver disorders at high frequencies at clinical test stages, accordingly, the development of these agents for clinical practice has been very difficult.
Arteriosclerosis is a disease essentially involving unique features of hypertrophy of inner vascular membrane and lipid accumulation. Recent research works indicate that suppression of macrophage foaming essentially functioning for the formation of arteriosclerotic lesions possibly degenerates arteriosclerotic lesions. Macrophage-derived foam cell (storing cholesterol ester as lipid droplet inside the cell) is observed in arteriosclerotic lesions. It is indicated that macrophage foaming is deeply involved in the progress of the disease. Additionally, it is reported that ACAT activity is elevated in the wall of blood tubes in arteriosclerotic lesions, indicating that cholesterol ester is accumulated in the wall of blood tubes (Gyres, P. J. et al., Exp. Mole. Pathol., 44, 329-339 (1986)).
Due to the inhibition of cholesterol esterification by ACAT inhibitors, free cholesterol is generated inside cells and is then eliminated with high-density lipoprotein (HDL) to be transferred to and metabolized in liver (reverse transfer by HDL). It is suggested that the accumulation of cholesterol in diseased sites is thereby suppressed. Consequently, direct anti-arteriosclerotic action is exerted. A report tells that ACAT includes two sub-types, namely an ACAT type present in small intestine and an ACAT type, present in vascular wall (Quinoonen, P. M. et al., Biochem., 27, 7344-7350 (1988)). Conventional research works on ACAT inhibitors have mostly been carried out by using the ACAT type present in small intestine and liver (Tomoda, H. et al., J. Antibiotics 47, 148-153 (1994)). Based on the assumption that a pharmaceutical agent selectively inhibiting the ACAT type which presents in vascular wall may work as a therapeutic agent of arteriosclerosis with less side effects, the present inventors have synthetically produced such inhibitors and have carried out examinations on them.
So as to attain the object, the inventors have made investigations. Consequently, the inventors have found that a compound represented by the general formula I: 
represents a divalent residue of benzene with a substituent(s), heterocycle-condensed benzene which may or may not have a substituent, pyridine which may or may not have a substituent, cyclohexane or naphthalene or 
Ar represents an aryl group which may or may not have a substituent;
X represents xe2x80x94NHxe2x80x94, oxygen atom or sulfur atom;
Y represents xe2x80x94NR4xe2x80x94, oxygen atom, sulfur atom, sulfoxide or sulfone;
Z represents single bond or xe2x80x94NR5xe2x80x94;
R4 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent;
R5 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent; and
n represents an integer of 0 to 15;
a salt thereof or a solvated compound thereof exerts an excellent ACAT inhibitory action. Thus, the invention has been achieved.
The inventors have found that the inventive compounds exert ACAT inhibitory actions in an organ-specific manner and an action inhibiting the transfer of intra-cellular cholesterol and that the inventive compounds are particularly useful as anti-hyperlipidemia agents with an excellent action to reduce cholesterol in blood and as a prophylactic and therapeutic agent of arteriosclerosis with an action to suppress macrophage foaming.
Thus, the compound represented by the general formula I, a salt thereof or a solvated product thereof is provided in accordance with the invention.
Additionally, the invention provides pharmaceutical compositions comprising the compound represented by the general formula I, a salt thereof or a solvated product thereof, together with carriers pharmaceutically acceptable.
Still additionally, the invention provides the compounds shown as the above formula I, salts thereof or solvated compounds thereof, and ACAT inhibitors, agents inhibiting intra-cellular cholesterol transfer, blood cholesterol-reducing agents, or macrophage foaming-suppressing agents. In other words, the invention provides therapeutic and prophylactic agents of diseases including hyperlipidemia, arteriosclerosis, arteriosclerosis of carotid and cerebral arteries, cerebrovascular diseases, ischemic cardiac diseases, coronary arteriosclerosis, nephrosclerosis, arteriosclerotic nephrosclerosis, arteriocapillary sclerotic nephrosclerosis, malignant nephrosclerosis, ischemic intestinal diseases, acute mesenteric blood tube occlusion, chronic intestinal angina, ischemic colitis, aortic aneurysm and occlusive arteriosclerosis (ASO).
As compounds similar to the compound of the formula I, 3-(benzothiazol-2-ylthio)-N-(phenyl)propanamide and 3-(benzoxazol-2-ylthio)-N-(phenyl)propanamide are disclosed in J. Chem. Eng. Data, 27, 207 (1982) and Fungitsidy, Ed. Melnilov, N. N. Izd. Fan Uzb. SSR: Tashkent, USSR. 82-88 (1980), respectively. However, it has absolutely never been known that these compounds exert ACAT inhibitory actions.
Preferable examples of the compound represented by the general formula I in accordance with the invention include a compound represented by the following formula II, a salt thereof or a solvated product thereof, and a compound represented by the following formula III, a salt thereof or a solvated product thereof: 
represents a divalent residue of benzene with a substituent(s), heterocycle-condensed benzene which may or may not have a substituent, pyridine which may or may not have a substituent, cyclohexane or naphthalene or 
X represents xe2x80x94NHxe2x80x94, oxygen atom or sulfur atom;
Y represents xe2x80x94NR4xe2x80x94, oxygen atom, sulfur atom, sulfoxide or sulfone;
Z represents single bond or xe2x80x94NR5xe2x80x94;
R1, R2 and R3 may be the same or different and represent hydrogen atom, a lower alkyl group, a lower alkoxyl group, halogen atom, hydroxyl group, phosphate group, sulfonamide group, or amino group which may or may not have a substituent; otherwise, any combination of two of R1, R2 and R3 represents an alkylene dioxy group;
R4 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent;
R5 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent; and
n represents an integer of 0 to 15; 
wherein
X represents xe2x80x94NHxe2x80x94, oxygen atom or sulfur atom;
Y represents xe2x80x94NR4xe2x80x94, oxygen atom, sulfur atom, sulfoxide or sulfone;
Z represents single bond or xe2x80x94NR5xe2x80x94;
R1, R2 and R3 may be the same or different and represent hydrogen atom, a lower alkyl group, a lower alkoxyl group, halogen atom, hydroxyl group, phosphate group, sulfonamide group, or amino group which may or may not have a substituent; otherwise, any combination of two of R1, R2 and R3 represents alkylene dioxy group;
R4 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent;
R6, R7 and R8 may be the same or different and represent hydrogen atom, a lower alkyl group which may or may not have a substituent, a lower alkoxyl group which may or may not have a substituent, halogen atom, hydroxyl group, carboxyl group, an alkoxycarbonyl group which may or may not have a substituent, an alkylcarbonyloxy group which may or may not have a substituent, an alkylcarbonyl group which may or may not have a substituent, carbamoyl group which may or may not have a substituent, a hydroxyalkyl group, phosphate group, cyano group, nitro group, sulfonamide group, amino group which may or may not have a substituent, an aminoalkyl group which may or may not have a substituent, or a heterocyclic residue; otherwise, any combination of two of R6, R7 and R8 represents an alkylene dioxy group, provided that R6, R7 and R8 never simultaneously represent hydrogen atom; and
n represents an integer of 0 to 15. More preferable is a compound represented by the following general formula IV, a salt thereof or a solvated product thereof: 
wherein
represents 
X represents xe2x80x94NHxe2x80x94, oxygen atom or sulfur atom;
Y represents xe2x80x94NR4xe2x80x94, oxygen atom, sulfur atom, sulfoxide or sulfone;
Z represents single bond or xe2x80x94NR5xe2x80x94;
R1, R2 and R3 may be the same or different and represent hydrogen atom, a lower alkyl group, a lower alkoxyl group, halogen atom, hydroxyl group, phosphate group, sulfonamide group, or amino group which may or may not have a substituent; otherwise, any combination of two of R1, R2 and R3 represents an alkylene dioxy group;
R4 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent;
R5 represents hydrogen atom, a lower alkyl group, an aryl group or a silylated lower alkyl group which may or may not have a substituent;
R9, R10, R9xe2x80x2, R10xe2x80x2, R9xe2x80x3, R10xe2x80x3, R9xe2x80x2xe2x80x3, and R10xe2x80x2xe2x80x3 may be the same or different and represent hydrogen atom, a lower alkyl group which may or may not have a substituent, a lower alkoxyl group which may or may not have a substituent, halogen atom, hydroxyl group, carboxyl group, an alkoxycarbonyl group which may or may not have a substituent, an alkylcarbonyloxy group which may or may not have a substituent, an alkylcarbonyl group which may or may not have a substituent, carbamoyl group which may or may not have a substituent, a hydroxyalkyl group, phosphate group, sulfonamide group, amino group which may or may not have a substituent, an aminoalkyl group which may or may not have a substituent, or a heterocyclic residue; otherwise, any combination of two thereof represents an alkylene dioxy group; and
n represents an integer of 0 to 15.
Ar in the general formula I represents an aryl group which may or may not have a substituent; a specifically preferable group is the following group: 
wherein R1, R2 and R3may be the same or different and represent hydrogen atom, a lower alkyl group, a lower alkoxyl group, halogen atom, hydroxyl group, phosphate group, sulfonamide group, or amino group which may or may not have a substituent; otherwise, any combination of two of R1, R2 and R3 represents an alkylene dioxy group.
The following group in the general formula I 
represents a divalent residue of benzene with a substituent(s), heterocycle-condensed benzene which may or may not have a substituent, pyridine which may or may not have a substituent, cyclohexane or naphthalene, or 
A divalent residue of benzene with a substituent preferably includes the group represented by the following formula: 
wherein R6, R7, and R8 may be the same or different and represent hydrogen atom, a lower alkyl group which may or may not have a substituent, a lower alkoxyl group which may or may not have a substituent, halogen atom, hydroxyl group, carboxyl group, an alkoxycarbonyl group which may or may not have a substituent, an alkylcarbonyloxy group which may or may not have a substituent, an alkylcarbonyl group which may or may not have a substituent, carbamoyl group which may or may not have a substituent, a hydroxyalkyl group, phosphate group, cyano group, nitro group, sulfonamide group, amino group which may or may not have a substituent, an aminoalkyl group which may or may not have a substituent, or a heterocyclic residue; otherwise, any combination of two of R6, R7, and R8 represents alkylene dioxy group, provided that R6, R7, and R8 never simultaneously represent hydrogen atom.
A divalent residue of heterocycle-condensed benzene which may or may not have a substituent preferably includes the group represented by the following formula: 
wherein R6 and R7 may be the same or different and represent those described above; the ring B represents a saturated or unsaturated heterocyclic group with at least one oxygen atom, nitrogen atom or sulfur atom in a 5- to 7-membered ring condensed with benzene ring.
A divalent residue of pyridine preferably includes the group represented by the formula: 
wherein R9, R10, R9xe2x80x2, R10xe2x80x2, R9xe2x80x3, R10xe2x80x3, R9xe2x80x2xe2x80x3, and R10xe2x80x2xe2x80x3 may be the same or different and represent hydrogen atom, a lower alkyl group which may or may not have a substituent, a lower alkoxyl group which may or may not have a substituent, halogen atom, hydroxyl group, carboxyl group, an alkoxycarbonyl group which may or may not have a substituent, an alkylcarbonyloxy group which may or may not have a substituent, an alkylcarbonyl group which may or may not have a substituent, carbamoyl group which may or may not have a substituent, a hydroxyalkyl group, phosphate group, sulfonamide group, amino group which may or may not have a substituent, an aminoalkyl group which may or may not have a substituent, or a heterocyclic residue; otherwise, any combination of two thereof represents an alkylene dioxy group.
The aryl group in R4 and R5 is preferably phenyl group, naphthyl group and the like; and these aryl groups may or may not have the substituents described above.
The lower alkyl group represented by each symbol in the general formula I preferably includes a linear or branched alkyl group with one to 15 carbon atoms, preferably one to 10 carbon atoms and more preferably one to 6 carbon atoms. For example, the lower alkyl group is preferably methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, tert-butyl group, n-pentyl group, n-hexyl group or the like. As the lower alkoxyl group, preference is given to an alkoxyl group comprising alkyl groups such as those described above. The alkoxycarbonyl group preferably contains the alkoxyl groups described above. The alkylcarbonyloxy group preferably contains the lower alkyl groups described above. The alkylcarbonyl group preferably contains the lower alkyl groups described above.
Additionally, the lower alkyl groups, the lower alkoxyl groups, the alkoxycarbonyl groups, the alkylcarbonyloxy groups, the alkylcarbonyl groups or the carbamoyl groups may or may not have substituents. These substituents may work as alternative substituents for each other; for example, alkoxy lower alkyl group, alkoxyalkoxyl group, lower alkoxyalkoxycarbonyl groups, alkoxycarbonyl-substituted alkyl group, alkoxycarbonyl-substituted alkoxyl group, alkoxycarbonyl-substituted alkoxycarbonyl group may be possible.
Still additionally, other substituents include for example halogen atoms such as chlorine atom and fluorine atom, hydroxyl group, silyl groups such as trimethylsilyl group, dimethyl t-butylsilyl group, and dimethylphenylsilyl group, saturated or unsaturated heterocyclic residues containing one or two or more oxygen atoms, nitrogen atoms or sulfur atoms in the ring thereof, such as oxethanyl group, tetrahydrofuryl group and pyrrolidinyl group. The alkylene dioxy group preferably contains a linear or branched alkylene group with one to 6 carbon atoms.
The halogen atom is preferably fluorine atom, chlorine atom, bromine atom or iodine atom or the like. The amino group may or may not be substituted with one or two substituents. The substituents for the amino group preferably include lower alkyl groups such as those described above, aryl groups such as phenyl group and naphthyl group, and aralkyl groups such as benzyl group and phenethyl group; and the aromatic rings thereof may or may not be substituted with lower alkyl groups and lower alkoxyl groups such as those described above, additionally. Furthermore, two of the substituents for the amino group together may form a 5- to 7-membered ring which may or may not contain oxygen, sulfur or nitrogen. The heterocyclic residues are preferably of a monocycle, a polycycle or a condensed ring comprising a 5- to 7-membered ring, saturated or unsaturated, containing one or two hetero-atoms, preferably one to four hetero-atoms such as oxygen atom, nitrogen atom or sulfur atom; and these heterocyclic residues may or may not be substituted with the lower alkyl groups, the lower alkoxyl groups, the alkylene dioxy groups, halogen atom, the amino group, and substituted amino groups. The heterocyclic residues include for example tetrazolyl group, 2-, 4- or 5-imidazolyl group, 3- or 4-pyrazolyl group, 2-, 4- or 5-oxazolyl group, 2-, 4- or 5-thiazolyl group, oxazolin-2-, 4- or 5-yl group, [1,3]-dioxylan-2- or 4-yl group, and these heterocyclic residues substituted with lower alkyl groups such as methyl group and ethyl group.
The acid addition salt of the compound I in accordance with the invention includes for example salts thereof with inorganic acids, such as hydrochloride salt, sulfate salt, nitrate salt, and phosphate salt, and salts thereof with organic acids, such as methanesulfonate salt, maleate salt, fumarate salt and citrate salt.
Additionally, the solvated product thereof is prepared by adding solvents used for the production and purification thereof, for example water and alcohol, to the compound I, with no specific limitation, as long as the solvated product never disadvantageously affects the ACAT inhibitory action. The solvated product is preferably a hydrated product thereof.
The invention relates to the compound represented by the general formulae I, II, III or IV, a salt thereof or a solvated product thereof, and a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier. More specifically, the invention relates to a pharmaceutical composition as ACAT inhibitor, intra-cellular cholesterol transfer inhibitory agent, blood cholesterol-reducing agent, or macrophage foaming-suppressing agent. Still furthermore, the invention relates to a pharmaceutical composition as a prophylactic and therapeutic agent of hyperlipidemia, arteriosclerosis, cerebrovascular diseases, ischemic cardiac disorders, ischemic colon disorders or aortic aneurysm.
Still additionally, the invention relates to a method for therapeutically treating diseases due to ACAT, intra-cellular cholesterol transfer, blood cholesterol or macrophage foaming and a method for therapeutically treating hyperlipidemia, arteriosclerosis, cerebrovascular diseases, ischemic cardiac disorders, ischemic colon disorders or aortic aneurysm, comprising administering a therapeutically effective dose of the compound represented by the general formula I, II, III or IV, a salt thereof or a solvated product thereof.
Still more additionally, the invention relates to the use of the compound represented by the general formula I, II, III or IV, a salt thereof or a solvated compound thereof for producing an ACAT inhibitor, an agent inhibiting intra-cellular cholesterol transfer, an agent reducing blood cholesterol, or an agent suppressing macrophage foaming and the use thereof for therapeutically treating hyperlipidemia, arteriosclerosis, cerebrovascular diseases, ischemic cardiac disorders, ischemic colon disorders or aortic aneurysm.
The compound I can be produced by a variety of known methods, with no specific limitation. The compound I can be produced for example by the following steps.
1. Process of Producing Compound with Z Representing Single Bond
(1) Following the reaction scheme described hereinbelow, reaction of carboxylic acid represented by the general formula V or a reactive derivative thereof, for example acid halide, with amine represented by the general formula VI generates an amide derivative represented by the general formula VII. Reaction of the resulting compound represented by the general formula VII with a compound represented by the general formula VIII can generate the objective compound Ixe2x80x2 with Z representing single bond: 
wherein R11 represents an elimination group; and R12 represents a residue of a reactive derivative of hydroxyl group or carboxyl group.
A general method for peptide synthesis is applicable to the reaction of the compound V with the compound VI. R11 in the general formula V is preferably halogen atom such as chlorine atom and bromine atom; and the residue of a reactive derivative as represented by R12 is preferably an acid anhydride residue of mesyl acid, tosyl acid, acetic acid and pivalic acid. For example, the two compounds react together in the presence of a condensing agent in a solvent whereby the objective compound can be recovered. As the condensing agent, use may be made of for example 1-(3xe2x80x2-dimethylaminopropyl)-3-ethylcarbodiimide (WSC) and 1,3-dicyclohexylcarbodiimide (DCC), singly or in combination with 1-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide (HOSu). As the solvent, use can be made of for example dimethylformamide, methylene chloride, chloroform, tetrahydrofuran and toluene, singly or in combination thereof, with no specific limitation.
The reaction varies, depending on the raw materials to be used; the reaction proceeds generally at 0 to 100xc2x0 C., preferably around ambient temperature, for one to 30 hours, preferably 10 to 20 hours. When a highly reactive carboxylic halogenide is used as the compound V, the compound V is allowed to react with the compound VI in the presence of bases, for example triethylamine, 4-dimethylaminopyridine or N-methylmorpholine by general methods.
The starting compounds V and VI are known compounds; the compound V can be produced by a method comprising oxidizing haloalkyl alcohol with Jones reagents and the like, while the compound VI can be produced by a method comprising subjecting a nitrobenzene derivative to a reductive reaction including contact reduction to prepare a corresponding aniline derivative.
The reaction of the compound VII thus recovered by the aforementioned methods with the compound VIII can be progressed in the presence or absence of bases in a solvent. As the solvent, use may be made of those described above, including bases for example inorganic bases including alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate and potassium carbonate, and alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; and organic bases such as pyridine, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, and N,N-dimethylaniline.
(2) Following the reaction represented by the following scheme, the compound represented by the general formula VIII is allowed to react with free carboxylic acid or an inactive carboxylic acid form as the compound represented by the general formula V, to recover a carboxylate derivative represented by the general formula IX. The resulting compound represented by the general formula IX or a reactive derivative thereof, for example an acid halide, is allowed to react with an aniline derivative represented by the general formula VI, to generate the objective compound Ixe2x80x2 with Z representing single bond: 
wherein R11 represents an elimination group and R12 represents a residue of a reactive derivative of hydroxyl group or carboxyl group.
The reaction of the compound VIII with the compound V can be facilitated by the second step described above in (1). The reaction is progressed by using potassium hydroxide as the base and ethanol as the solvent, preferably. The reaction of the compound VI with the compound III can be facilitated by the first step described above in (1). If necessary, R12 in the compound IX can be modified as a reactive derivative residue, prior to the reaction.
2. Process of Producing Compound Ixe2x80x3 with Z Representing xe2x80x94NHxe2x80x94.
The compound represented by the general formula I wherein Z represents xe2x80x94NHxe2x80x94 can be produced by a variety of methods. The compound can be produced by the method represented by the following reaction scheme. 
By allowing an isocyanate derivative represented by the general formula X to react with an aniline derivative represented by the general formula VI, a urea derivative represented by the general formula XI can be recovered.
By allowing the compound VIII to react with the resulting urea derivative, the objective compound Ixe2x80x3 with Z representing xe2x80x94NHxe2x80x94 can be recovered.
As regards the reaction of the compound X with the compound VI at the first step, reaction of one to 2 equivalents of the compound VI with one equivalent of the compound X in a solvent can yield the compound XI. As the solvent, preferably, use is made of for example methylene chloride, chloroform, ether, tetrahydrofuran, toluene, xylene, and dimethylformamide, with no specific limitation. The reaction progresses at 0xc2x0 C. to the boiling point of the solvent used, over one to 24 hours.
The isocyanate derivative represented by the general formula X is a known compound and can be produced, for example by a method comprising allowing the carboxylic acid as the compound X to react with diphenylphosphoryl azide in the presence of a base (the method by Shioiri et al.) and a method en route of acid azide prepared by allowing an acid halide as the compound V to react with sodium azide.
The reaction of the compound XI with the compound VIII can be facilitated according to the second step of the reaction 1.1.
The intermediates and objective compounds as recovered in the individual reactions can be isolated and purified by purification methods routinely used in synthetic organic chemistry, for example filtration, extraction, rinsing, drying, concentration, recrystallization, and various chromatographic means. Furthermore, the intermediates can be subjected, with no purification, to next reaction.
The resulting compound I can be modified as an acid addition salt in a conventional manner.
Alternatively, solvated products thereof with solvents such as reaction solvent and recrystallization solvent, specifically hydrated product thereof, may also be recovered.
Specific examples of the compounds recovered by the production methods are shown in Tables 1, 2, 3, 4, 5, 6, 7 and 8.
The inventive compound represented by the general formula I has an ACAT inhibitory action and/or an action inhibiting intra-cellular cholesterol transfer and is therefore useful as a therapeutic agent of hyperlipidemia or a therapeutic agent of arteriosclerosis in the field of clinical medicine. Particularly because the inventive compound exerts an action selectively inhibiting an ACAT type present in vascular wall, the inventive compound possibly exerts less side effects, compared with non-selective ACAT inhibitors, which is preferable as an effective ingredient of pharmaceutical agent.
The inventive pharmaceutical composition contains the compound represented by the general formula I, an acid addition salt thereof or a solvated product thereof as the effective ingredient. Singly or in combination with other pharmaceutically acceptable carriers such as excipients, binders and diluents, the effective ingredient can be prepared as dosage forms such as tablet, capsule, granule, powder, injection and suppository. These dosage forms can be produced according to known methods. For preparing an oral dosage form, the compound represented by the general formula I is formulated with an appropriate combination of excipients such as starch, mannitol and lactose, binders such as sodium carboxymethylcellulose and hydroxypropylcellulose, disintegrators such as crystal cellulose and carboxymethylcellulose, lubricants such as talc and magnesium stearate, and fluidity-enhancing agents such as light silicic anhydride.
The inventive pharmaceutical composition is administered orally or parenterally.
The dose of the inventive pharmaceutical composition varies, depending on the body weight, age, sex and diseased conditions of a patient. For an adult, generally, the compound represented by the general formula I is preferably administered at 1 to 1000 mg, preferably 5 to 200 mg per day in one to three dividend doses.
The ACAT inhibitory action of the inventive compound represented by the general formula I is tested in the following experimental examples.
In a conventional manner, microsome was prepared from the thoracic aorta of a rabbit fed with a 1% cholesterol diet for 8 weeks, which was then suspended in 0.15 M phosphate buffer, pH 7.4, to prepare an enzyme solution. An enzyme solution was prepared from a rabbit small intestine on normal diet, which was defined as an enzyme solution derived from small intestine.
The ACAT inhibitory activity was assayed by a modification of the method by J. G. Hyder, J. Lipid Res., 24, 1127-1134 (1983). More specifically, 2 xcexcl of a test compound dissolved in dimethyl sulfoxide (DMSO) was added to 88 xcexcl of 0.15 M phosphate buffer, pH 7.4 containing 14C-Oleoyl-CoA (40 xcexcM, 60,000 dpm) and 2.4 mg/ml bovine serum albumin, for incubation at 37xc2x0 C. for 5 minutes. 10 xcexcl of an enzyme solution was added to the resulting solution for reaction at 37xc2x0 C. for 5 days (small intestine-derived enzyme solution was subjected to reaction for 3 minutes). Subsequently, the reaction was terminated by adding 3 ml of chloroform/methanol (2/1) and 0.5 ml of 0.04 N hydrochloric acid to the reaction solution, to extract lipid. The solvent layer was concentrated and dried, which was then dissolved in hexane and spotted on a TLC plate (manufactured by Merck, Co.). The plate was eluted with hexane:ether:acetic acid (75:25:1). The radioactivity of the resulting cholesterol ester fraction was assayed by BAS 2000 (manufactured by Fuji Photo Film, Co., Ltd.). Compared with the radioactivity of a control prepared by single addition of DMSO, IC50 was determined. The results are shown in Table 9.
J774 cells or HepG2 cells were inoculated on a 24-well plate; J774 cells and HepG2 cells were cultured in DMEM and MEM culture broths (each of the broths containing 10% calf fetus serum), respectively, in a 5% CO2 incubator at 37xc2x0 C. for 24 hours. These culture broths were individually exchanged to 0.5 ml of DMEM and MEM containing 10 xcexcg/ml 25-OH cholesterol and a test sample, for 18-hr culturing. After discarding the culture media, the resulting cultures were rinsed twice with PBS and extracted with 1.5 ml of hexane:isopropanol (3:2), for concentration and drying. The extracts were dissolved in isopropanol containing 0.2 ml of 10% Triton X-100, to assay total cholesterol (TC) and free cholesterol (FC) by using Cholesterol E Test WAKO (manufactured by Wako Pure Chemicals, Co.) and Free Cholesterol E Test Wako (manufactured by Wako Pure Chemicals, Co.). After extraction, cellular residue was solubilized in 0.25 ml of 2 N NaOH at 37xc2x0 C. for 30 minutes, to assay protein by BCA Protein Assay Reagent (Pierce). Based on the difference between TC and FC, cholesterol ester was calculated per protein, to determine IC50, compared with the calculated control IC50 value. The results are shown in Table 10.
The following compounds were tested as control compounds by the same method. The results are shown in Tables 7 and 8.
Control 1:
5-[2-(2-(4-fluorophenyl)ethyl)-3-(1-methyl-1H-imidazol-2-yl)-2H-1-benzopyran-6-yl]oxy-2,2-dimethyl-N-(2,6-diisopropylphenyl)pentanamide (WO92/09582)
Control 2:
(+)-(S)-2-[5-(3,5-dimethylpyrazol-1-yl)pentasulfinyl]-4,5-diphenylimidazole (EP, A, 523941)
Control 3:
N-(2,2,5,5-tetramethyl-1,3-dioxan-4-ylcarbonyl)-xcex2-alanine 2(S)-[Nxe2x80x2-(2,2-dimethylpropyl)-Nxe2x80x2-nonylureido]-1(S)-cyclohexyl ester (EP, A, 421441)
Control 4:
[5-(4,5-diphenyl-1H-imidazol-2-ylthio)pentyl]-N-heptyl-2-benzoxazolamine (WO93/23392)